Sex steroid signaling in the lung (particularly estrogens) is of considerable interest, given epidemiological data showing more asthma in pre-menopausal women and aging men, making this of importance in both sexes. However, a fundamental issue is that while studies in immune cells and some animal studies suggest estrogens enhance inflammation per se other studies find that estrogens blunt asthma symptoms, and furthermore blunt airway reactivity. These conflicting data reflect the complexity of estrogen signaling that cannot be explained by effects on the immune system alone, and could involve perhaps opposite effects on structural cells of the airway. In this regard, what is also not clear is whether specific estrogen receptors (ERs) play differential roles that might explain these contrasts. The overall goal is to understand ER expression and signaling in structural airway cells in the context of inflammation and asthma, using airway smooth muscle (ASM) as a model. In asthma, inflammation enhances ASM contractility, proliferation and extracellular matrix (ECM) production. We find that human ASM from both sexes expresses ER? and ER but not G-protein coupled GPR30. While estrogens can non-genomically reduce ASM [Ca2+]i, what is not known are: A) Effects of inflammation or asthma on ER expression and functionality; B) Roles of ER? vs. ER in inflamed/asthmatic ASM, particularly genomic actions likely more relevant in vivo. Preliminary studies in human ASM show: 1) Asthmatic or cytokine-exposed ASM from both sexes express more ER than ER?; 2) With inflammation, ER functionality is enhanced compared to ER?, helping reduce [Ca2+]i, ASM proliferation and ECM at physiological estradiol levels; 3) ER? and ER signaling diverge in inflamed ASM, with differential effects on cAMP vs. NF?B and p38. In vivo, in a mixed allergen mouse model with a Th2 immune profile, ASM ER is increased (less so in epithelium), while ER KO results in greater airway thickening, reactivity, and ASM expression of Ca2+ regulatory and ECM proteins. Conversely, ER-specific agonists blunt inflammation, airway reactivity and remodeling, and ASM expression of ECM proteins due to mixed allergens. Thus, our overall hypothesis is that inflammation leads to a differential and greater role for ER in ASM, and that ER can blunt contractility and remodeling. These concepts will be explored via the following Specific Aims. Aim 1: To determine mechanisms by which inflammation alters ER expression profile in human ASM; Aim 2: To determine the enhanced and differential role of ER in human ASM contractility with inflammation/asthma; Aim 3: To determine mechanisms by which ER? vs. ER blunt human airway remodeling; Aim 4: In a mouse model of allergic asthma, to determine the effect of ER? vs. ER on structural and functional airway changes. In Aims 1-3, we will use human ASM tissues from 30-50y old non-asthmatic and asthmatic males and females to examine mechanisms by which inflammation/asthma alters ER? vs. ER expression (Aim 1; focusing on NF?B, MAPK and STAT6), and mechanisms by which ER? vs. ER reduce [Ca2+]i/contractility (Aim 2) and remodeling (proliferation, ECM; Aim 3), focusing on the differential roles of cAMP/PKA/CREB vs. NF?B and p38. In Aim 4, a mixed allergen model will be applied to ER? vs. ER KO mice with/without ovariectomy and with/without THC or DPN (specific agonists) treatment. Structural, functional and signaling changes in ASM relevant to ER? vs. ER will be evaluated. The clinical significance lies in understanding how sex steroids influence structural airway cells in inflammation and asthma, overall helping to appreciate the mechanistic bases of the conflicting data regarding estrogens and asthma.